Summary/Abstract: Heterotrimeric G proteins are the major intracellular enzymes that receive signals from plasma membrane G protein coupled receptors (GPCRs) and transduce them to a broad array of intracellular effectors. Our lab has contributed much to the understanding of the function of a pair of G protein regulators, Ric-8A and Ric-8B. We identified Ric-8A and Ric-8B as G protein ? subunit binding proteins and showed that they possess the ability to accelerate the rate at which G? subunits release GDP and bind GTP. This activity is similar to GPCR action towards G protein heterotrimers. GPCRs stimulate G protein GDP/GTP exchange for the purpose of activating the G proteins to transmit signals. We have yet to find conclusive evidence that Ric-8 proteins facilitate G? GDP/GTP exchange for signaling purposes. We did find that deletion of the Ric-8A or Ric-8B genes in mice caused embryonic lethality. Culture of Ric-8A or Ric-8B knockout embryonic stem cells that were attained from viable embryos prior to death demonstrated that the cells had dramatically reduced levels of G proteins. This prompted our investigation into the role that Ric-8 proteins have in regulating G protein abundance. We found that Ric-8 proteins are molecular chaperones that facilitate protein folding of newly made G? subunits. When G proteins are made in cells lacking Ric-8 proteins, they are misfolded and rapidly degraded. We reconcile the in vitro GDP/GTP exchange stimulatory activity of Ric-8 with the folding function by proposing that Ric-8 proteins bind the intermediate of the in vitro exchange reaction in cells, newly-synthesized, nucleotide-free G proteins to facilitate first time GTP binding. Until now, all evidence indicated that Ric-8 proteins acted constitutively to fold G proteins. The premise of this new application is based on our recent data that show that Ric-8 activities are subject to dramatic regulation by post-translational phosphorylation. We will investigate a new link between mitogenic oncogene stimulus that leads to Ric-8 deregulation (dephosphorylation) and possible remodeling of cellular G protein levels. We have made important strides in our long-time collaborative efforts to investigate the structural basis by which Ric-8 proteins regulate G proteins. The work in this application will define the way that phosphorylation regulates Ric-8 function.